S-nitrosylation (SNO), the covalent addition of nitric oxide (NO) to a thiol, is a reversible modification with pervasive roles in physiology and pathophysiology. Cellular SNO signaling requires coordination of the enzymes that generate, sense, and degrade NO. These include isoforms of nitric oxide synthases, which are often overexpressed in tumors, as well as dedicated enzymes to denitrosylate and transnitrosylate SNOs. S- nitrosylation of cysteine thiols in the proteome has well-established consequences for signal transduction though low molecular weight SNOs (LWM-SNOs) remain poorly characterized. Considerable evidence implicates altered S-nitrosylation in cancer progression and metastasis with deficiency in a protein denitrosylase, S-nitrosoglutathione reductase (GSNOR), increasing likelihood of hepatocarcinoma presentation in mouse models and decreased GSNOR expression found in patient tumors. While numerous cellular SNOs have been identified, new technologies are needed to facilitate SNO analysis in order to uncover the molecular details of SNO signaling in cancer. Current analytical methodologies are largely indirect, unable to preserve labile S-nitrosylated cysteines in vivo and prone to chemical artifacts. As a result, the bona fide SNO targets of GSNOR have remained uncharacterized for nearly a decade after the discovery of the enzyme due to technological limitations. The goal of this proposal is to develop, apply, and validate a structurally novel probe to chemically trap SNOs in vivo in living cells and tissues in order to make global molecular analysis of the S-nitrosoproteome and metabolome more physiologically- and clinically-relevant. This technological innovation will be enabling within the burgeoning field of SNO biology by ensuring the validity of SNO targets identified, minimizing artifacts, enhancing storage of SNO samples for analysis, and being compatible with a wide array of analytical modalities for imaging and quantitation. Aim 1 will generate a stable and water soluble probe with selective reactivity towards SNOs. Aim 2 will validate that the probe quantitatively labels SNOs in cancer cells and will benchmark probe signal with an orthogonal quantitative measurement of SNO levels (ozone-based chemiluminescence) in parallel on the same samples. Aim 3 will evaluate the ability of the new probe design to detect altered SNO levels upon hypoxia, a hallmark of solid tumors, and organism-wide after in vivo SNO trapping in GSNOR knockout mice.